Rapid and prolonged response of oligodendrocyte lineage cells in standard acute cuprizone demyelination model revealed by in situ hybridization.

Dietary administration of a copper chelator, cuprizone (CPZ), has long been reported to induce intense and reproducible demyelination of several brain structures such as the corpus callosum. Despite the widespread use of CPZ as an animal model for demyelinating diseases such as multiple sclerosis (MS), the mechanism by which it induces demyelination and then allows robust remyelination is still unclear. An intensive mapping of the cell dynamics of oligodendrocyte (OL) lineage during the de- and remyelination course would be particularly important for a deeper understanding of this model. Here, using a panel of OL lineage cell markers as in situ hybridization (ISH) probes, including Pdgfra, Plp, Mbp, Mog, Enpp6, combined with immunofluorescence staining of CC1, SOX10, we provide a detailed dynamic profile of OL lineage cells during the entire course of the model from 1, 2, 3.5 days, 1, 2, 3, 4,5 weeks of CPZ treatment, as well as after 1, 2, 3, 4 weeks of recovery from CPZ treatment. The result showed an unexpected early death of mature OLs and response of OL progenitor cells (OPCs) in vivo upon CPZ challenge, and a prolonged upregulation of myelin-forming OLs compared to the intact control even 4 weeks after CPZ withdrawal. These data may serve as a basic reference system for future studies of the effects of any intervention on de- and remyelination using the CPZ model, and imply the need to optimize the timing windows for the introduction of pro-remyelination therapies in demyelinating diseases such as MS.

Botulinum toxin type A inhibits M1 macrophage polarization by deactivation of JAK2/STAT1 and IκB/NFκB pathway and contributes to scar alleviation in aseptic skin wound healing.

The non-neuronal and non-muscular effects of botulinum toxin type A (BTXA) on scar reduction has been discovered. This study was designed to investigate the effects of BTXA on macrophages polarization during the early stage of skin repair. A skin defect model was established on the dorsal skin of SD rats. BTXA was intracutaneous injected into the edge of wound immediately as the model was established. Histological examinations were performed on scar samples. Raw 264.7 was selected as the cell model of recruited circulating macrophages, and was induced for M1 polarization by LPS. Identify the signaling pathways that primarily regulated M1 polarization and respond to BTXA treatment. Application of BTXA at early stage of injury significantly reduced the scar diameter without delaying wound closure. BTXA treatment improved fiber proliferation and arrangement, and inhibited angiogenesis in scar granular tissue. The number of M1 macrophages and the levels of pro-inflammation were decreased after treated with BTXA in scar tissues. LPS activated JAK2/STAT1 and IκB/NFκB pathways were downregulated by BTXA, as well as LPS induced M1 polarization. At early stage of skin wound healing, injection of BTXA effectively reduced the number of M1 macrophages and the levels of pro-inflammatory mediators which contributes to scar alleviation. BTXA resisted the M1 polarization of macrophages induced by LPS via deactivating the JAK2/STAT1 and IκB/NFκB pathways.

The self-management experiences of individuals who perceive health as beyond their control: An interpretive phenomenological study of individuals with ischemic heart disease.

This qualitative study aimed to gain an understanding of what it means to live with ischemic heart disease for individuals who perceive health as beyond their control and how these individuals navigate their choices regarding adhering or not adhering to self-management behavior. Participants were recruited through purposive sampling, and semi-structured interviews were conducted. Content analysis was employed to identify themes and subthemes in the interview data. The theme, "attribution of ischemic heart disease," revealed that the participants attributed their condition to lifestyle, critical events, and the natural aging process. The theme, "experiences of self-management," highlighted the different behaviors among participants who perceived health to be beyond their control. The theme, "barriers and facilitators," identified factors such as a strong sense of responsibility toward family members, the work environment, and access to medical resources. Our study showed that despite perceiving their health to be beyond their control, some individuals may still adhere to self-management practices. Understanding factors such as "attribution" and "barriers and facilitators" can provide nurses with insights into the patients' decisions to adhere or not adhere to self-management behaviors.

Establishment of a Beagle Dog Model of Dry Eye Disease.

Purpose: To establish a Beagle dog model of dry eye disease (DED). Methods: DED models were induced by surgical removal of orbital lacrimal glands and entire resection of third eyelids in the left eyes of six Beagle dogs. Intact right eyes served as self-controls. Non-anaesthetized Schirmer test (STT), tear break-up time (TBUT), and fluorescein staining grading were performed monthly after operation. Interleukin (IL)-1ß, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) levels were detected in tears and conjunctiva tissues. Six months after surgery, conjunctiva and cornea were collected and histopathologically analyzed. Results: Signs of DED appeared within one month after surgery and then remained stable. STT values were significantly reduced by 88% within 3 weeks after operation and remained stable over months with 1.6 ± 0.4 mm. Mean TBUT decreased significantly within two months after operation and maintained 5.2 ± 1.1 seconds. The mean fluorescein staining score was highest at the first month and then was reduced, eventually reaching a balance with 11.0 ± 1.3 points. Elevated levels of IL-1ß, IL-8, IL-10, and TNF-α were detected in tears and conjunctivas of operated eyes. Hematoxylin and eosin staining showed cornea neovascularization in the corneal stroma with thickened stroma layer and disorganized collagen bundles. Periodic acid-Schiff staining revealed a reduced function of conjunctival goblet cells. Conclusions: A combined type of DED model on the Beagle dog was established by removal of the orbital lacrimal gland and resection of the third eyelid. This DED model is easily accessible and is stable at six-month observation. Translational Relevance: The surgery-induced Beagle dog DED model is easily accessible and stable over a relatively long time.

Increased M2 Isoform of Pyruvate Kinase in Fibroblasts Contributes to the Growth, Aggressiveness, and Osteoclastogenesis of Odontogenic Keratocysts.

To investigate the role of glycolysis and the M2 isoform of pyruvate kinase (PKM2) in odontogenic keratocysts (OKCs), the glycolytic flux of primary odontogenic keratocyst fibroblasts (OKC-Fs) and normal oral mucosa fibroblasts (OM-Fs) was determined by glucose uptake, lactate production, and cell proliferation assays. Wound healing assay and Matrigel-coated chamber system were used to investigate the effects of PKM2 on migration and invasion capacities of OKC-Fs. Co-culture of OKC-Fs with osteoclast precursors (RAW264.7 cells) was used to clarify the role of glycolysis in the osteoclastogenic effects of OKC-Fs. In addition, hypoxia-inducible factor 1α and some key enzymes related to glycolysis, including PKM2, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A, were detected to assess the activation of glycolysis in OKC stroma by immunohistochemistry. Results showed that the glucose uptake and lactate production were significantly higher in OKC-Fs than OM-Fs. PKM2 was elevated in OKC-Fs compared with that in OM-Fs. PKM2 significantly regulated glycolysis, proliferation, migration, invasion, and osteoclastogenic effects of OKC-Fs. Additionally hypoxia-inducible factor 1α, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A were markedly overexpressed in OKC stroma, and correlated with PKM2. Moreover, the expression of PKM2 was regulated by oxygen concentration in vitro. In sum, PKM2-mediated glycolysis regulated the growth, aggressiveness, and osteoclastogenesis of OKC.

Pyruvate Kinase M2 Mediates Glycolysis in the Lymphatic Endothelial Cells and Promotes the Progression of Lymphatic Malformations.

Metabolism plays a pivotal role in the formation of the lymphatic vasculature. Pyruvate kinase M2 (PKM2) is typically a metabolic marker of proliferating cells and maintains the growth of vascular endothelial cells. In this study, the potential status of PKM2 in lymphatic endothelial cells and the pathogenesis of lymphatic malformations (LMs) was investigated. The glycolysis index, including glucose uptake, ATP, and lactate production, stayed at a relatively high level in human dermal lymphatic endothelial cells (HDLECs) compared with human umbilical vein endothelial cells, whereas the inhibition of PKM2 by shikonin or PKM2 knockdown significantly suppressed glycolysis, migration, tubular formation, and invasion of HDLECs. Moreover, compared with lymphatic vessels in healthy skin, lymphatic vessels of LMs expressed PKM2 highly, and this expression correlated with infection of LMs. Meanwhile, the overexpression of PKM2 in HDLECs strengthened the proliferation, migration, tubular formation, and invasion of HDLECs. The findings from further experiments in a rat LM model support that targeting PKM2 by shikonin significantly impedes the progression of LMs, even in an infected LM rat model. Taken together, these results indicate that PKM2 plays a pivotal role in the activation of LECs and promotes the progression of LMs, whereas the inhibition of PKM2 can effectively suppress the pathogenesis of LM lesions in the rat model.

The YAP signaling pathway promotes the progression of lymphatic malformations through the activation of lymphatic endothelial cells.

BACKGROUND: To investigate whether the YAP/TAZ (Yes-associated protein/transcriptional coactivator with PDZ binding motif) pathway contributes to the pathogenesis of lymphatic malformations (LMs). METHODS: YAP, TAZ, CTGF (connective tissue growth factor), and Ki-67 were detected in LMs by immunohistochemistry. The colocalization of YAP and Ki-67 was analyzed by double immunofluorescence. Pearson's correlation and cluster analyses were performed to analyze the relationships between these proteins. Human dermal lymphatic endothelial cells (HDLECs) were used for mechanistic investigation. Rat models of LMs were established to investigate the role of the YAP pathway in LM development. RESULTS: Compared with those in normal skin, the expression levels of YAP, TAZ, CTGF, and Ki-67 were significantly upregulated in lymphatic endothelial cells (LECs) of LMs. Interestingly, YAP and CTGF presented much higher expression levels in infected LMs. In experiments in vitro, lipopolysaccharide (LPS) enhanced the expression of YAP in a concentration- and time-dependent manner via the increased phosphorylation of Erk1/2 (extracellular signal-regulated kinase 1/2). Moreover, the proliferation, invasion, and tubule formation of HDLECs increased significantly in accordance with the activation of the YAP signaling pathway. Furthermore, LM rat models validated that LPS facilitated the development of LMs, which was dependent on the activation of YAP. CONCLUSIONS: The data reveal that activation of the YAP signaling pathway in LECs may play a crucial role in the progression of LMs. IMPACT: Compared with that in normal skin, the YAP signaling pathway was activated in LECs of LMs. Inhibiting the YAP signaling pathway attenuated the proliferation, invasion, and tubule formation of HDLECs. Additionally, the activation of the YAP signaling pathway could promote LM development in a rat model. Activation of the YAP signaling pathway in LECs may play a crucial role in the progression of LMs. The YAP signaling pathway was activated in LMs. Inhibition of the YAP signaling pathway could promote regression of the lesions.

Bleomycin: A novel osteogenesis inhibitor of dental follicle cells via a TGF-ß1/SMAD7/RUNX2 pathway.

BACKGROUND AND PURPOSE: Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown. EXPERIMENTAL APPROACH: Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-ß1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function. KEY RESULTS: Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-ß1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-ß1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin- or TGF-ß1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-ß1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-ß1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin. CONCLUSION AND IMPLICATIONS: SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-ß1 inhibited osteogenic differentiation of DFCs via a TGF-ß1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth.

Hydrostatin-SN10 Ameliorates Pancreatitis-Induced Lung Injury by Affecting IL-6-Induced JAK2/STAT3-Associated Inflammation and Oxidative Stress.

Hydrostatin-SN1 (peptide sequence, DEQHLETELHTLTSVLTANGFQ), a kind of peptides extracted from snake venom, has been reported to have anti-inflammatory effect, but its truncated mutant hydrostatin-SN10 (peptide sequence, DEQHLETELH) on pancreatitis-induced acute lung injury has not been well documented. Interleukin- (IL-) 6-induced Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway is involved with inflammatory and oxidative stress activities and may be associated with the pathogenesis of lung injury, and related molecules were measured. Taurocholate-induced pancreatitis associated with acute lung injury was established and treated with hydrostatin-SN10. Pancreatitis was confirmed by measuring the serum levels of amylase, lipase, and trypsinogen and urinary amylase. Lung injury was determined by histologically assessing acinar cell changes. The related molecules of IL-6-induced JAK2/STAT3-associated inflammation and oxidative stress were quantitated by real time-PCR, Western blot, and/or immunochemical assay. Hydrostatin-SN10 reduced the levels of serum amylase, lipase, and trypsinogen and urinary amylase when compared with the model group (p < 0.05). Hydrostatin-SN10 significantly inhibited the IL-6-stimulated JAK2/STAT3 pathway and reduced the number of apoptotic cells via the downregulation of caspase 3 and BAX (proapoptotic) and upregulation of Bcl2 (antiapoptotic) (p < 0.05). IL-6 induced the increase in the levels of JAK2 and STAT3, which was reversed by hydrostatin-SN10 treatment (p < 0.05). In addition, hydrostatin-SN10 reduced the expression of IL-6 and TNF- (tumor necrosis factor-) α and increased the level of IL-10 (p < 0.05). On the other hand, hydrostatin-SN10 treatment increased the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) and the levels of malondialdehyde (MDA) and alanine aminotransferase (ALT) (p < 0.05). These results suggest that hydrostatin-SN10 may inhibit pancreatitis-induced acute lung injury by affecting IL-6-mediated JAK2/STAT3 pathway-associated inflammation and oxidative stress.

Elevated ATF4 Expression in Odontogenic Keratocysts Epithelia: Potential Involvement in Tissue Hypoxia and Stromal M2 Macrophage Infiltration.

The aim of this study was to investigate the expression of the activating transcription factor 4 (ATF4) in odontogenic keratocysts (OKC), its association with hypoxia and M2-polarized macrophages infiltration, and its potential relationships with angiogenesis in OKC. The expression of ATF4, hypoxia-inducible factor 1α (HIF-1α), macrophage colony-stimulating factor (M-CSF), and receptor activator of nuclear factor κ-B ligand (RANKL) in OKC samples and normal oral mucosa (OM) was detected by immunohistochemistry. Meanwhile, microvessel density (MVD) was measured using antibody against CD31. M2-polarized macrophages were identified using double-staining for CD68+ and CD163+. The correlations of ATF4 with HIF-1α, M-CSF, and M2-polarized macrophages infiltration were determined by Spearman's rank correlation test and hierarchical clustering. Human immortalized oral epithelial cells (HIOECs) were used in in vitro experiments. Our data showed that the expression of HIF-1α, ATF4, and M-CSF was significantly upregulated in the epithelium of OKC when compared with the OM. The expression of ATF4 was positively correlated with that of HIF-1α, M-CSF, MVD, and M2-polarized macrophages infiltration. Elevated expression of ATF4 in the epithelial lining of OKC may facilitate the M2 macrophages infiltration in response to hypoxia, leading to the development of OKC.

Clonal diversity of Ureaplasma species and its relationship with oligozoospermia and semen quality in Chinese infertile males.

Whether Ureaplasma spp. are a causative agent of male infertility remains controversial. Previous studies concerning Ureaplasma spp. and male infertility have been confined to the species level of Ureaplasma. Currently, an expanded multilocus sequence typing (eMLST) scheme has been established with high discriminatory power. The aim of this study was to use eMLST to explore the distribution of Ureaplasma spp. and to analyze its role in oligozoospermia and semen quality. A total of 480 semen samples were obtained from Chinese infertile males. The associations between Ureaplasma spp. with oligozoospermia and semen characteristics were further evaluated. Phylogenetic analysis revealed that 102 Ureaplasma spp. could be separated into two clusters and seven sub-groups. Within cluster I (U. parvum), eST16 and eST41 were the most frequent clones. For cluster II (U. urealyticum), eST82 and eST147 were the most prevalent clones. Sub-groups A and C belonging to cluster I and sub-group 1 belonging to cluster II showed an association with oligozoospermia, in contrast with the Ureaplasma spp. negative group (P < 0.05). Compared with the negative group, semen motility decreased in sub-group 2, especially for non-progressive motility (P < 0.05). These results indicated that sub-groups A and C belonging to cluster I (U. parvum) and sub-group 1 belonging to cluster II (U. urealyticum) were shown to be associated with oligozoospermia. Sub-group 2 belonging to cluster II may have the ability to impair semen motility, especially for non-progressive motility.

Biochanin A partially restores the activity of ofloxacin and ciprofloxacin against topoisomerase IV mutation-associated fluoroquinolone-resistant Ureaplasma species.

PURPOSE: This study aims to investigate the synergistic antimicrobial activity of four phytoalexins in combination with fluoroquinolones against Ureaplasma spp., a genus of cell wall-free bacteria that are intrinsically resistant to many available antibiotics, making treatment inherently difficult. METHODOLOGY: A total of 22 958 urogenital tract specimens were assessed for Ureaplasma spp. identification and antimicrobial susceptibility. From these, 31 epidemiologically unrelated strains were randomly selected for antimicrobial susceptibility testing to determine the minimum inhibitory concentration (MIC) of four fluoroquinolones and the corresponding quinolone resistance-determining regions (QRDRs). Synergistic effects between fluoroquinolones and four phytoalexins (reserpine, piperine, carvacrol and biochanin A) were evaluated by fractional inhibitory concentration indices (FICIs). RESULTS: Analysis of the QRDRs suggested a vital role for the mutation of Ser-83→Leu in ParC in fluoroquinolone-resistant strains, and the occurrence of mutations in QRDRs showed significant associations with the breakpoint of levofloxacin. Moreover, diverse synergistic effects of the four phytoalexins with ofloxacin or ciprofloxacin were observed and biochanin A was able to enhance the antimicrobial activity of fluoroquinolones significantly. CONCLUSION: This is the first report of the antimicrobial activity of biochanin A in combination with fluoroquinolones against a pathogenic mycoplasma, and opens up the possibility of using components of biochanin A as a promising therapeutic option for treating antibiotic-resistant Ureaplasma spp. infections.

Effects of germination on the nutritional properties, phenolic profiles, and antioxidant activities of buckwheat.

Germination is considered to be an effective process for improving the nutritional quality and functionality of cereals. In this study, changes of nutritional ingredients, antinutritional components, chemical composition, and antioxidant activities of buckwheat seeds over 72 h of germination were investigated, and the reasons for these changes are discussed. With the prolonged germination time, the contents of crude protein, reducing sugar, total phenolics, total flavonoids, and condensed tannins increased significantly, while the levels of crude fat, phytic acid, and the activity of trypsin inhibitor decreased. Phenolic compounds, such as rutin, vitexin, isovitexin, orientin, isoorientin, chlorogenic acid, trans-3-hydroxycinnamic acid, and p-hydroxybenzoic acid increased significantly during the germination process, which may be due to the activation of phenylalanine ammonialyase. The improvement of flavonoids led to significant enhancement of the antioxidant activities of germinated buckwheat. Germinated buckwheat had better nutritional value and antioxidant activities than ungerminated buckwheat, and it represented an excellent natural source of flavonoids and phenolic compounds, especially rutin and C-glycosylflavones. Therefore, germinated buckwheat could be used as a promising functional food for health promotion.

Effect of black soybean extract on the suppression of the proliferation of human AGS gastric cancer cells via the induction of apoptosis.

Black soybean is known to have a health-promoting effect because of its high content of polyphenolic compounds and antioxidant activities. The objective of the present study was to investigate the chemopreventive effects of black soybean extract against human AGS gastric cancer cells and its possible mechanism in inducing apoptosis. Black soybean extract was obtained by extracting black soybean with acidified aqueous acetone, and its phytochemical constituents, as determined by HPLC-DAD methods, were demonstrated to contain various phenolics. The black soybean extract inhibited AGS cell growth in a dose-dependent manner with an IC(50) of 3.69 mg/mL as measured by the MTT assay. This growth inhibition effect was further confirmed by the CFDA-SE assay. Flow cytometry analysis showed that black soybean extract dose-dependently induced apoptosis of AGS cells. Moreover, the involvement of black soybean extract in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, caspase-3, and PARP. The results of the present study indicated that black soybean extract could be used as an apoptosis inducer in AGS cells and a natural chemopreventive agent in the treatment of human gastric cancer.

Antioxidant activity and phenolic compositions of lentil (Lens culinaris var. Morton) extract and its fractions.

Phenolic compounds were extracted from Morton lentils using acidified aqueous acetone. The crude Morton extract (CME) was applied onto a macroresin column and desorbed by aqueous methanol to obtain a semipurified Morton extract (SPME). The SPME was further fractionated over a Sephadex LH-20 column into five main fractions (I-V). The phytochemical contents such as total phenolic content (TPC), total flavonoid content (TFC), and condensed tannin content (CTC) of the CME, SPME, and its fractions were examined by colorimetric methods. Antioxidant activity of extracts and fractions were screened by DPPH scavenging activity, Trolox equivalent antioxidant capacity (TEAC), ferric reduced antioxidant power (FRAP), and oxygen radical absorbing capacity (ORAC) methods. In addition, the compositions of active fractions were determined by HPLC-DAD and HPLC-MS methods. Results showed that the fraction enriched in condensed tannins (fraction V) exhibited significantly higher values of TPC, CTC, and antioxidant activity as compared to the crude extract, SPME, and low molecular weight fractions (I-IV). Eighteen compounds existed in those fractions, and 17 were tentatively identified by UV and MS spectra. HPLC-MS analysis revealed fraction II contained mainly kaempferol glycoside, fractions III and IV mainly contained flavonoid glycosides, and fraction V was composed of condensed tannins. The results suggested that the extract of Morton lentils is a promising source of antioxidant phenolics and may be used as a dietary supplement for health promotion.

Comparative studies on the chemical and cell-based antioxidant activities and antitumor cell proliferation properties of soy milk manufactured by conventional and commercial UHT methods.

The aims of this work were to compare antiproliferation, antioxidant activities and total phytochemicals and individual isoflavone profiles in soy milk processed by various methods including traditional stove cooking, direct steam injection, direct ultrahigh temperature (UHT), indirect UHT, and a two-stage simulated industry method, and a selected commercial soy milk product. Various processing methods significantly affected total saponin, phytic acid, and total phenolic content and individual isoflavone distribution. The laboratory UHT and the two-stage processed soy milk exhibited relatively higher total phenolic content, total flavonoid content, saponin and phytic acid than those processed by the traditional and steam processed methods. Thermal processing caused obvious intertransformation but did not cause severe degradation except for breaking down of aglycons. Thermal processing significantly increased antioxidant capacities of soy milk determined by chemical analyses, but decreased cellular antioxidant capacities as compared to the raw soy milk. The raw and all processed soy milk exhibited antipoliferative activities against human HL-60 leukemia cells, AGS gastric tumor cells, and DU145 prostate cancer cells in a dose-dependent manner. The raw soy milk, but not the processed soy milk, exhibited a dose-dependent antiproliferative effect against colorectal adenocarcinoma Caco-2 cells. Taken together, these results indicate that various thermal processing methods change not only phytochemcials but also potential health-promoting effects of soy milk.

Protective effects of a flavonoid-rich extract of Hypericum perforatum L. against hydrogen peroxide-induced apoptosis in PC12 cells.

Hypericum perforatum L. has been used traditionally as an antidepressant for the treatment of mild to moderate depression. In a previous study, a flavonoid-rich extract of Hypericum perforatum L. (FEHP) was prepared and its antioxidant activity was determined by a series of models in vitro. In the present study, the protective effects of FEHP against hydrogen peroxide-induced apoptosis in rat pheochromocytoma line PC12 cells were investigated by MTT assay, lactate dehydrogenase (LDH) release assay, flow cytometry analysis and DNA fragmentation assay. Following a 4 h exposure of PC12 cells to H2O2, a significant decrease in the cell viability and increased levels of LDH release were observed. However, pretreatment of PC12 cells with FEHP prior to H2O2 exposure elevated the cell viability, decreased the levels of LDH release and decreased the occurrence of apoptotic cells. Also, the intensity of H2O2-induced DNA laddering was inhibited in a dose-dependent fashion by a DNA fragmentation assay. These results suggested that FEHP possessed protective effects against H2O2-induced apoptosis in PC12 cells and FEHP might be useful in the treatment of oxidative stress-related neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

Antioxidant effects of phlorotannins isolated from Ishige okamurae in free radical mediated oxidative systems.

Three phlorotannins, including phloroglucinol, diphlorethohydroxycarmalol, and 6,6'-bieckol, were isolated from Ishige okamurae by column chromatography. The structures of the phlorotannins were determined on the basis of spectroscopic analysis, including NMR and mass spectrometry (MS) techniques. Antioxidant effects of phlorotannins were measured by direct free radical scavenging activities using the electron spin resonance spectrometry (ESR) technique and cellular systems in vitro. The results indicated that diphlorethohydroxycarmalol and 6,6'-bieckol showed potential radical scavenging activities against the 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, alkyl, and superoxide radicals. Moreover, no cytotoxicities of the phlorotannins on human fetal lung fibroblasts cell line (MRC-5), mouse macrophages cell line (RAW264.7), and human leukemic cell line (HL-60) were observed. In addition, diphlorethohydroxycarmalol and 6,6'-bieckol significantly reduced the intracellular reactive oxygen species level assessed by 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay in RAW264.7 cells, and myeloperoxide (MPO) activity in HL-60 cells and radical-mediated oxidation of cell membrane proteins in RAW264.7 cells were dose-dependently inhibited in the presence of diphlorethohydroxycarmalol and 6,6'-bieckol. In conclusion, these results suggested that phlorotannins could be used as novel functional foodstuffs or antioxidants in the cosmetic and drug industries.

Hypocholesterolemic effects of a flavonoid-rich extract of Hypericum perforatum L. in rats fed a cholesterol-rich diet.

In a previous study, a flavonoid-rich extract of Hypericum perforatum L. (FEHP) was prepared and its antioxidant activity was determined by a series of models in vitro. In this study, the hypocholesterolemic effects of FEHP in rats fed a cholesterol-rich diet were tested. Forty Wistar rats fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks were used. The serum lipid levels, as well as malondialdehyde (MDA) and activity of superoxide dismutase (SOD) and catalase (CAT) in serum and liver, were examined. Cholesterol-rich diet induced hypercholesterolemia was manifested in the elevation of serum lipid levels such as total cholesterol (TC), total triglycerides (TG), and low density lipoprotein cholesterol (LDL-C). Administration of middle-dose (75 mg/kg of BW/day) and high-dose (150 mg/kg of BW/day) FEHP significantly lowered the serum levels of TC, TG, and LDL-C, while increasing the serum level of high density lipoprotein cholesterol (HDL-C). Also, the content of MDA in serum and liver decreased significantly after oral administration of FEHP compared with those of rats fed a cholesterol-rich diet. In addition, FEHP increased the activity of SOD in serum and liver, but the activity of CAT was significantly elevated only in liver. These results suggested that the hypocholesterolemic effects of FEHP might be due to its abilities to lower serum TC, TG, and LDL-C levels as well as to slow the lipid peroxidation process and to enhance the antioxidant enzyme activity.